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Analysis of specificities of Importin-± from different organisms through structural and calorimetric techniques

Grant number: 17/10571-6
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): August 01, 2017
Effective date (End): December 31, 2018
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal Investigator:Marcos Roberto de Mattos Fontes
Grantee:Cíntia Akemi Fukuda
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Associated research grant:13/24705-3 - The filamentous fungus Neurospora crassa as a model organism for the functional characterization of proteins/transcription factors that regulate the carbohydrate metabolism, AP.TEM

Abstract

The communication between the cell nucleus and the cytoplasm happens through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. Among the known transport routes that enable the transport of macromolecules into or out of the nucleus, through the recognition of specific signaling sequences, the so-called Classical Import Nuclear Pathway is the most well characterized. In this way, the Importin-± (Imp±) protein acts on the identification of the proteins to be transported to the nucleus from the recognition of nuclear localization sequences (NLS). The structure of the Imp± protein has already been elucidated and characterized in some organisms. By analyzing the amino acid sequences of these proteins, it was possible to classify them into three subfamilies: ±1, ±2, and ±3. The differences between the Imp± proteins of each family show the specificities of these proteins in the recognition of NLSs, depending on the organism and function that they carry out. That is, the same NLS peptide may exhibit variations in affinity and binding mode when it interacts with Imp± from distinct families. The aim of this project is to compare the affinity and the binding mode of a same NLS peptide with proteins Imp± of families ±1 and ‘2. The NLSs that will be used in this project are transcription factors of the fungus Neurospora crassa and the results of this study may contribute to the identification of NLS specificities in the interaction with N. crassa Imp± (NcImp±). Initially, the affinities of the interactions of the same fungal NLS peptide with NcImp± (family ±1) and Imp± from Mus Musculus (MmImp±-family ±2) will be analyzed. Next, the NcImp± / NLS peptides and MmImp± / NLS peptides will be submitted to crystallization experiments and the best crystals subjected to X-ray diffraction, data collection and processing. After the modeling and recombination stages, the structures of the complexes will be elucidated and compared to verify structural specificities in the interaction of the same NLS peptide with Imp± from different families. In addition, in order to quantify the interactions between these NLS peptides and the MmImp± and NcImp±, isothermal titration calorimetry (ITC) experiments will be performed to provide the dissociation constant, stoichiometry, enthalpy and entropy of the interactions. The present project is linked to Thematic Project (proc.2013 / 24705-3). (AU)